Fig. 9: Images of bacterial samples acquired with Pocket MUSE.

Two different populations (Gram + and Gram-) of bacteria can be separated with Pocket MUSE. Aliquots of (a) deionized water, (b) an E. coli (Gram-) culture, (c) a B. subtilis (Gram + ) culture, and (d) a mixture of E. coli culture and B. subtilis culture imaged with Pocket MUSE. All samples were stained with 0.01% w/v DAPI and 0.02 % w/v WGA-AF594, and imaged under the same conditions. Images show (a) no bright spots, (b) blue-grayish spots, (c) a mixture of reddish and orangish spots, and (d) a mixture of blue-grayish, reddish and orangish spots dispersed across the FOV. For each image, a close-up view of the region labeled with the box (box size: 250 × 250 µm²) is shown below. For (b)–(d), the center pixels of three bright spots in each image, pointed to by the arrows, are shown in the boxes below. The RGB values (top to bottom) of the pixels are labeled on the side. The nucleic acid in E. coli (DAPI stain) contributes to the blue-grayish speckles (R ≈ G ≈ B). Peptidoglycan on the B. subtilis surface (WGA-AF594 stain) contributes to the reddish speckles (R > G + B). Nucleic-acid-rich endospores in B. subtilis contribute to the orangish speckles (R > G, R > B & R < G + B). e–h bivariate histogram representation of the bright pixels in (a)–(d), showing the distribution of colors. All images were median-filtered and background-corrected before analysis. Only bright pixels are counted (e.g., four times greater than the mean value of the corresponding image). X-axises represent the ratio between red and blue channels. Y-axises represent the ratio between red and green channels. Scale bars: 300 µm.